Site-specific protein lipoylation analysis <i>via</i> aldehyde-acetal probe labelling and reversible enrichment (DA-Lipo)

Protein lipoylation regulates metabolic processes closely related to cancer and neurodegeneration, but only five human lipoylated proteins have been identified, underscoring the need for robust analytical methods. We present DA-Lipo, a one-step labelling/enrichment strategy for site-specific lipoylation analysis. Terephthalaldehyde monodiethylacetal selectively tags lipoyl-peptides <i>via</i> cyclic thiol-aldehyde chemistry, enabling reversible enrichment and MS detection with a diagnostic immonium ion (419.18 Da). DA-Lipo confirmed known sites and revealed a novel lipoylation in GSTP2, offering a robust tool for functional studies.